The inability to effectively transform organisms through integration of desirable genetic information into the genome of cells is a significant limitation to genetic research, acting as a bottleneck to the otherwise rapidly developing field of biotechnology. A June 1997, Scientific American article "Overcoming the Obstacles of Gene Therapy" by Theodore Friedmann, focuses on the current limitations of cellular genetic transformation which predominantly arise from the inability to effectively administer genetic transformation vectors into cells. The article addresses the limitations of cellular genetic transformation as applied to medicine, indicating that, by overcoming these obstacles, gene therapy, or the admission of specific genes to cells for the purpose of treating disfunction, has the potential to revolutionize medical science. The National Institutes of Health (NIH) have recognized the potential of gene therapy and have given it priority among their areas of scientific interest.
The genetic transformation of economically significant crops is also a major biotechnology research thrust area. The ability to transform crops for improved productivity, enhanced quality, resistance to climate, pests, and herbicides, and extended climatic range is an important pursuit in providing for an expanding world population.
Cellular transformation is important to many other industrial sectors. Production of pharmaceuticals through transformation biotechnology is an emerging focus area. Plant, mammalian, and bacterial colonies are genetically transformed to produce desired pharmaceuticals and pharmaceutical precursors. Chemical producing bioreactors using transformed organisms as production units are also emerging as a significant component of this industrial sector.
Current methods for cellular transformation focus on the delivery and genomic incorporation of transformation vectors to target cells. These genetic vectors may take a variety of forms, but are typically double stranded or single stranded "naked" DNA sequences or bacterially derived, plasmid vectors. The latter are short (typically 2--10 kbase) circular DNA sequences which contain complete gene sequences, promoters, enhancers, restriction sites to enable further genetic manipulation of the plasmid, and other advantageous segments such as reporter genes for indication of when a transformation event has occurred. Introduction of these vectors into a cell, through the cell boundary, may result in transient expression of the inserted genes, and may ultimately stably transform the target cell by insertion of the delivered gene sequences into the host genome.
Delivery of transformation vectors to cells is accomplished in a variety of ways. The effectiveness of each method varies widely with cell type. For dicotyledonous plant cells, effective vector delivery may be performed using an agrobacterium vector, which infects the cells of the host plant, and subsequently delivers plasmid DNA to the infected cells. This method has limited application to various stages of growth of dicotyledonous cells, and is significantly limited in application to monocotyledonous plants, including the agronomically significant cereals. This latter limitation arises because few monocotyledonous plants are natural hosts for agrobacteria. Therefore, recent research activity has focused upon alternative methods for transforming this sector of the plant kingdom.
Similar viral transformation techniques exist for mammalian cells, with an underlying limitation that most of these infectious techniques are specific to certain cell types, and fundamentally lack applicability to a wider group of cell types.
A variety of mechanical methods have been developed to overcome the specificity of infectious transformation techniques. These methods, not being limited to infectious pathways, are designed to provide transformation techniques to a wide array of cell types. Methods include electroporation, microinjection, DNA application into the budding stage of flowering plants, laser- or opto-poration, microprojectile bombardment and whisker-mediated transformation. Essentially, each of these methods employs a mechanical means to damage the cell and its cellular boundary to facilitate the introduction of foreign DNA into the cell. In each of these methods, efficiency is often very low, and often the gene expression is only transiently manifested. Further, these methods are not ideally suitable for continuous-processing systems. Electroporation, employs a, or a series of, electrical pulses to (typically) a cellular suspension, which creates transient, electrically-induced pores in the cell membrane through which vectors may be introduced. Microinjection employs a precisely controlled microscale needle to puncture the cellular membrane and deliver vectors into the cell. Laser-or opto-poration creates chemical, electrical and thermal gradients around a target cell, opening the cell and facilitating the transport of genetic material into the cell. Microprojectile bombardment utilizes a variety of methods to ballistically fire vector-coated particles through cellular boundaries, to facilitate either directly or indirectly, the introduction of vectors into the cell. For example, Chang et al have developed a technique for delivering plant transformation vectors using microprojectile bombardment. In their technique, small metal particles on the order of 0.5-5 .mu.m are coated with plasmid vectors and accelerated via various methods through the cellular boundary. Those that remain within the host cell shed their DNA coating, that in turn may be transcribed for expression of delivered genes. This delivery mechanism, although well suited to laboratory studies, is recognized as being highly inefficient, and is currently unsuitable for continuous-feed bioreactor implementation. One of the problems with this method is that although in some instances, the coated vectors are delivered to a cell and may promote transformation of that cell, often, the microprojectile travels through the cell without effective plasmid delivery. Further, the assault of the projectile is often too damaging for the cell to recover, resulting in cell death.
A fundamental limitation of existing transformation methodologies is the inability to precisely control genetic vector delivery, and the associated damage created by the method. For instance, optimal electroporation protocols are a fine balance between effective plasmid delivery, and the destruction or loss of viability of cells due to excessive damage during the electroporation procedure. The pores created are often too large and cannot be resealed; or fail to close quickly enough to prevent excessive influx of surrounding media into the cells, resulting in cell swelling and death. Similarly, laser- or opto-poration is highly damaging, with the fundamental focal limitation of light being too large to produce acceptably small pores. Further, laser excitation produces a columnar destruction path which is not limited to the boundary of the target cell. Along this destruction path, heat and the production of free radicals may destroy the cell nucleus, surrounding plasmid vectors, and ultimately the cell itself. The damage of microprojectile bombardment is also fundamentally uncontrolled. Ballistic particles may excessively disrupt both cell internal structures as well as cellular boundaries, destroying viability of cells along their destructive path.
Slow processing rate, low transformation efficiency, lack of general applicability, and excessive damage limit the effectiveness of all present cellular transformation mechanisms. Current methodologies rely upon either natural biological mechanisms or coarse, mechanically-based means of administering genetic material into target cells. Mechanically based methods are extremely damaging and result in very low transformation rates, and are quite costly. The biological methodologies, employing bacterial or viral infection as the transformation mechanism (i.e. transfection), are only narrowly applicable, working selectively on organisms that act as hosts to the infective vector.
In 1985, Perez et al indicated in Radiation Research that ionizing and ultraviolet irradiation of rodent cells increases the efficiency of stable transformation of these cells. The mechanism of this increase in efficiency is the creation of radiation-induced lesions within the genomic DNA of these cells to provide sites for incorporation of transformation vectors thereby increasing the probability of stable transfection. Perez et al's methodology uses unregulated radiation to create lesions in the genomic DNA to facilitate incorporation at these lesion sites of previously delivered genetic material. Perez et al do not address delivery of material into cells at all. Particularly, Perez et al do not employ energy-regulated radiation to create localized stress sites within the cellular boundary to facilitate genetic material into the cell. Moreover, Perez et al's methodology intentionally damages the cell, including the contained genomic DNA, to facilitate genomic incorporation of previously delivered genetic material.
Traditional discussions of linear energy transfer (LET) and relative biological effects (RBE) of alpha-particles and other ionizing radiations focus on the potential for ionizing radiation to damage the functionality of a cell through directly ionizing effects and the generation of free-radicals which in turn damage the internals of a cell. Hall, incorporated herein by reference, indicates in Radiobiology for the Radiologist (4.sup.th ed., J. B. Lippincott Company 1994) that a LET of 100 keV/.mu.m is optimal in terms of producing a biological effect on a cell. Due to natural DNA repair mechanisms and the redundancy of the double helix, manipulation of or damage to chromosomal DNA is maximized when both strands of the DNA helix are damaged directly across from each other. 100 keV/.mu.m is maximally efficient for damaging both strands of the double helix, as this LET corresponds to ionizing events at roughly 2 nm intervals, the width of the double helix. Hall further indicates that " . . . much more densely ionizing radiation . . . will readily produce double strand breaks, but energy will be wasted because the ionizing events are too close together. `RBE is the ratio of doses to produce equal biological effect`" Thus, the most efficient LET to affect the entire cell (i.e. through chromosomal damage) is that which corresponds to 2 bond breakages in a 2 nm pathlength, or 100 keV/.mu.m.